Tick Salivary Proteins Research Articles

T-cell signaling pathways inhibited by the tick saliva immunosuppressor, Salp15

The Ixodes scapularis salivary protein Salp15 inhibits the activation of T cells through its interaction with the coreceptor CD4. Salp15 prevents the activation of Lck upon TCR engagement and the formation of lipid rafts. We have now analyzed the signaling pathways that are inhibited by the tick salivary protein in CD4(+) T cells. Salp15 affects tyrosine phosphorylation of several early signal components downstream of Lck, including LAT and Vav1, which results in improper actin polymerization. The effect of Salp15 is due to its interaction with CD4, as no effect was observed in CD4-negative T cells. Finally, we demonstrate that the peptide that mediates the interaction of Salp15 with CD4, P11, is able to recapitulate the immunosuppressive activity of the whole protein. These results clarify the molecular mechanisms of action of Salp15 on T cells and suggest that binding to CD4 is sufficient to elicit its immunosuppressive effect.

Anaplasma phagocytophilum subverts tick salivary gland proteins

Anaplasma phagocytophilum is a bacterium that is transmitted by Ixodes spp. ticks, in which it resides in salivary glands. Ticks inoculate the pathogen into hosts together with an array of salivary molecules that reduce host anti-tick inflammation. Sukumaran et al. recently showed that A. phagocytophilum uses a tick salivary protein, Salp16, to enhance its uptake from the host and into the salivary gland. Occupation and exploitation of tick salivary glands have implications for the maintenance and detection of A. phagocytophilum in its vector and early pathogen interactions with its hosts.

Taking advantage of saliva

The bacterium Anaplasma phagocytophilum uses a tick protein to help it set up camp in the insect's salivary glands, according to Sukumaran and colleagues (page 1507). Figure 1 A disease-causing bacterium uses a tick (shown) salivary protein to establish infection in the insect's salivary glands. Many tick-borne pathogens hijack host proteins to establish infection in their tick vector or mammalian host. Previous work by this group, for example, showed that the Lyme disease bacterium Borrelia burgdorferi induces the expression of the salivary protein Salp15 in infected Ixodes scapularis ticks as they feed. The bacterium then coats itself with Salp15 as it exits the tick, creating a shield against the destructive antibodies encountered in the mammalian host. Here, Sukumaran and colleagues used a similar approach to study the obligate intracellular bacterium A. phagocytophilum, which causes a common and sometimes deadly tick-borne disease in humans called anaplasmosis (formerly granulocytic ehrlichiosis). They found that if I. scapularis ticks fed on A. phagocytophilum–infected mice, the ticks selectively increased their expression of the salivary protein Salp16. But in this case, Salp16 was used for protection in the tick, not the mammalian host. Inhibiting Salp16 expression in the tick (using RNAi) reduced the number of bacteria in the insect's salivary glands, suggesting that Salp16 was needed for the bacterium to establish a foothold in the salivary gland where it resides after migrating from the gut. Once there, however, the bug no longer required Salp16 to maintain its residence. Inhibiting Salp16 had no effect on transmission of the bug to the mouse reservoir or on the initial uptake of the bug (with the blood meal) into the tick gut. The authors are now trying to determine how Salp16 helps A. phagocytophilum to colonize the salivary gland. In the meantime, blocking Salp16 in ticks might provide a way to disrupt the transmission cycle of the pathogen and thus lower the prevalence of disease.

An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands

Anaplasma phagocytophilum is the agent of human anaplasmosis, the second most common tick-borne illness in the United States. This pathogen, which is closely related to obligate intracellular organisms in the genera Rickettsia, Ehrlichia, and Anaplasma, persists in ticks and mammalian hosts; however, the mechanisms for survival in the arthropod are not known. We now show that A. phagocytophilum induces expression of the Ixodes scapularis salp16 gene in the arthropod salivary glands during vector engorgement. RNA interference–mediated silencing of salp16 gene expression interfered with the survival of A. phagocytophilum that entered ticks fed on A. phagocytophilum–infected mice. A. phagocytophilum migrated normally from A. phagocytophilum–infected mice to the gut of engorging salp16-deficient ticks, but up to 90% of the bacteria that entered the ticks were not able to successfully infect I. scapularis salivary glands. These data demonstrate the specific requirement of a pathogen for a tick salivary protein to persist within the arthropod and provide a paradigm for understanding how Rickettsia-like pathogens are maintained within vectors.

Antithrombotic properties of Ixolaris, a potent inhibitor of the extrinsic pathway of the coagulation cascade

Ixolaris is a two-Kunitz tick salivary gland protein identified in Ixodes scapularis that presents extensive sequence homology to TFPI. It binds to FXa or FX as scaffolds and inhibits tissue factor/FVIIa complex (extrinsic Xnase). Differently from TFPI, ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite, which may also results in decreased FXa activity into the prothrombinase complex. In this report, we show that recombinant (125)I-ixolaris interacts with rat and human FX in plasma and prolongs the prothrombin time (PT) and activated partial thromboplastin time (aPTT) in vitro. We have also investigated the effects of ixolaris in vivo, using a venous thrombosis model. Subcutaneous (s.c.) or intravenous (i.v.) administration of ixolaris in rats caused a dose-dependent reduction in thrombus formation, with complete inhibition attained at 20 microg/kg and 10 microg/kg, respectively. Antithrombotic effects were observed 3 h after s.c. administration of ixolaris and lasted for 24 h thereafter. Ex vivo experiments showed that ixolaris (up to 100 microg/kg) did not affect the aPTT, while the PT was increased by approximately 0.4-fold at the highest ixolaris concentration. Remarkably, effective antithrombotic doses of ixolaris (20 microg/kg) was not associated with bleeding which was significant only at higher doses of the anticoagulant (40 microg/kg). Our experiments demonstrate that ixolaris is an effective and possibly safe antithrombotic agent in vivo.

Tick modulation of the in-vitro expression of adhesion molecules by skin-derived endothelial cells

As a tick feeds, its saliva induces innate and acquired immune responses in the host, including leucocyte infiltration into the bite site. Tick salivary glands produce molecules, however, that counteract many host defences against blood feeding. The effects of salivary-gland extracts (SGE) of Dermacentor andersoni and Ixodes scapularis on the expression of various adhesion molecules [E-selectin, P-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)] by the sEND.1 cell line (which is based on cells from the subcutaneous tissue of mice) have now been investigated in vitro. The effects were found to differ with the tick species. The SGE of D. andersoni significantly down-regulated the expression of ICAM-1, whereas a similar extract prepared from I. scapularis significantly reduced the expression of P-selectin and VCAM-1. Tick salivary proteins therefore appear to have direct effects on adhesion-molecule expression, in addition to their previously established roles in down-regulating the pro-inflammatory cytokines that activate endothelial cells. It remains unclear exactly how the reduction of adhesion-molecule expression in the host's endothelial cells benefits the feeding tick but it may alter leucocyte migration to the bite site and/or reduce antigen presentation by the endothelial cells. It may also modulate the interactions between the host's leucocytes and any tick-borne pathogens, during initial infection of the endothelium.

The Lyme disease agent exploits a tick protein to infect the mammalian host

The Lyme disease agent, Borrelia burgdorferi, is maintained in a tick-mouse cycle. Here we show that B. burgdorferi usurps a tick salivary protein, Salp15 (ref. 3), to facilitate the infection of mice. The level of salp15 expression was selectively enhanced by the presence of B. burgdorferi in Ixodes scapularis, first indicating that spirochaetes might use Salp15 during transmission. Salp15 was then shown to adhere to the spirochaete, both in vitro and in vivo, and specifically interacted with B. burgdorferi outer surface protein C. The binding of Salp15 protected B. burgdorferi from antibody-mediated killing in vitro and provided spirochaetes with a marked advantage when they were inoculated into naive mice or animals previously infected with B. burgdorferi. Moreover, RNA interference-mediated repression of salp15 in I. scapularis drastically reduced the capacity of tick-borne spirochaetes to infect mice. These results show the capacity of a pathogen to use a secreted arthropod protein to help it colonize the mammalian host.

Identification of novel tick salivary gland proteins for vaccine development

Methods currently used to control Ixodes scapularis ticks rely principally on acaricidal applications which suffer from a number of limitations. Recently, host vaccination against ticks has been shown to be a promising alternative tick control method. In tick salivary glands, numerous genes are induced during the feeding process. Many of these newly expressed proteins are secreted in tick saliva and may play a role in modulating host immune responses and pathogen transmission. We have performed suppression subtraction hybridization to identify unique I. scapularis salary gland proteins specifically expressed during engorgement. We have cloned and sequenced ten unique salivary gland-associated cDNAs that are up-regulated during feeding. The protein products of these genes represent potential vaccine candidates for use in the control of ticks and to prevent transmission of tick-borne diseases.

Exploring tick saliva: from biochemistry to ‘sialomes’ and functional genomics

Tick saliva, a fluid once believed to be only relevant for lubrication of mouthparts and water balance, is now well known to be a cocktail of potent anti-haemostatic, anti-inflammatory and immunomodulatory molecules that helps these arthropods obtain a blood meal from their vertebrate hosts. The repertoire of pharmacologically active components in this cocktail is impressive as well as the number of targets they specifically affect. These salivary components change the physiology of the host at the bite site and, consequently, some pathogens transmitted by ticks take advantage of this change and become more infective. Tick salivary proteins have therefore become an attractive target to control tick-borne diseases. Recent advances in molecular biology, protein chemistry and computational biology are accelerating the isolation, sequencing and analysis of a large number of transcripts and proteins from the saliva of different ticks. Many of these newly isolated genes code for proteins with homologies to known proteins allowing identification or prediction of their function. However, most of these genes code for proteins with unknown functions therefore opening the road to functional genomic approaches to identify their biological activities and roles in blood feeding and hence, vaccine development to control tick-borne diseases.

A reassessment of argasid tick salivary gland ultrastructure from an immuno-cytochemical perspective

Previous morphological and histochemical studies of argasid tick salivary glands indicated that they were less complex than ixodid salivary glands, with only three granular cell types. The present study shows that there exist at least four different granular cell types in the salivary glands of the argasid tick Ornithodoros savignyi, based on immuno-localization of the anti-hemostatic factors, apyrase and savignygrin. Both anti-hemostatic factors were localized to dense core granule type 'a' and to granule type 'b', that shares a similar homogenous morphology with non-labeled granule type 'd'. Furthermore, the major tick salivary gland proteins (TSGPs), previously implicated in granule biogenesis, were localized to all the granular cell types. This indicates that granular cell types with different morphologies can express the same proteins, while cell types that show similar morphologies may not express the same proteins. Argasid tick salivary glands seem to be more complex than previously thought and might not be amenable to morphological classification alone. Alternative classification methodologies that rely on physical expression patterns of the salivary gland proteome might be more reliable as markers for a specific granular cell type.

Tick "talk": protein release by tick salivary cells.

<h2>Abstract</h2> Tick salivary gland proteins involved in exocytosis via soluble N-ethylmaleimide sensitive factor attachment proteins (SNAPs) and their receptor proteins (SNAREs) were recently identified. This new line of research looks at protein trafficking in the salivary glands. How do tick cells ‘talk' in order to move important proteins out of the granular acini and into the saliva?

The major tick salivary gland proteins and toxins from the soft tick, Ornithodoros savignyi, are part of the tick Lipocalin family: implications for the origins of tick toxicoses.

The origins of tick toxicoses remain a subject of controversy because no molecular data are yet available to study the evolution of tick-derived toxins. In this study we describe the molecular structure of toxins from the soft tick, Ornithodoros savignyi. The tick salivary gland proteins (TSGPs) are four highly abundant proteins proposed to play a role in salivary gland granule biogenesis of the soft tick O. savignyi, of which the toxins TSGP2 and TSGP4 are a part. They were assigned to the lipocalin family based on sequence similarity to known tick lipocalins. Several other tick lipocalins were also identified using Smith-Waterman database searches, bringing the tick lipocalin family up to 20. Phylogenetic analysis showed that most tick lipocalins group within genus-specific clades, suggesting that gene duplication and divergence of tick lipocalin function occurred after tick speciation, most probably during the evolution of a hematophagous lifestyle. TSGP2 and TSGP3 show high sequence identity and group terminal to moubatin, an inhibitor of collagen-induced platelet aggregation from the tick, O. moubata. However, no platelet aggregation inhibitory activity is associated with the TSGPs using ADP or collagen as agonists, suggesting that TSGP2 and TSGP3 duplicated after divergence of O. savignyi and O. moubata. This timing is supported by the absence of TSGP2-4 in the salivary gland extracts of O. moubata. The absence of TSGP2 and TSGP4 in salivary gland extracts from O. moubata correlates with the nontoxicity of this tick species. The implications of this study are that the various forms of tick toxicoses do not have a common origin, but must have evolved independently in those tick species that cause pathogenesis.

Identification of SNARE and cell trafficking regulatory proteins in the salivary glands of the lone star tick, Amblyomma americanum (L.)

Prostaglandin E 2 (PGE 2) stimulates secretion of tick salivary gland proteins via a phosphoinositide signaling pathway and mobilization of intracellular Ca 2+ ( Qian et al., 1998; Yuan et al., 2000). Highly conserved intracellular SNARE (soluble NSF attachment protein receptors) complex proteins are associated with the mechanism of protein secretion in vertebrate and invertebrate neuronal and non-neuronal cells. Proteins in the salivary glands of partially fed female lone star ticks cross-react individually with antibodies to synaptobrevin-2 (vesicle (v)-SNARE), syntaxin-1A, syntaxin-2 and SNAP-25 (target (t)-SNAREs), cytosolic α/β SNAP and NSF ( N-ethylmaleimide-sensitive fusion protein), Ca 2+ sensitive synaptotagmin, vesicle associated synaptophysin, and regulatory cell trafficking GTPases Rab3A and nSec1. V-SNARE and t-SNARE proteins form an SDS-resistant, boiling sensitive core complex in the salivary glands. Antibodies to SNARE complex proteins inhibit PGE 2-stimulated secretion of anticoagulant protein in permeabilized tick salivary glands. We conclude that SNARE and cell trafficking regulatory proteins are present and functioning in the process of PGE 2-stimulated Ca 2+ regulated protein secretion in tick salivary glands.

Identification of putative proteins involved in granule biogenesis of tick salivary glands.

Ticks secrete bioactive components during feeding that assist them in gaining a blood meal. Compounds secreted are stored in granules until a stimulus induces secretion during feeding. Biogenesis of tick secretory granules has not been investigated before. An adequate understanding of granule biogenesis could advance our understanding of tick salivary gland biology and could aid in the rational design of tick control methods. Putative tick salivary gland proteins 1-4 (TSGP1-4) involved in granule biogenesis were identified in this study based on their abundance in salivary gland extracts and granule preparations and their ability to aggregate under conditions of slight acidity and high calcium concentration. TSGP2 and TSGP3 have been identified as previously described toxic and nontoxic homologues, respectively, while toxicity was also associated with TSGP4.

Prostaglandin E 2-stimulated secretion of protein in the salivary glands of the lone star tick via a phosphoinositide signaling pathway

Previous studies identified a prostaglandin E 2 (PGE 2) receptor in the salivary glands of partially fed female lone star ticks, Amblyomma americanum (L.). In the present studies, protein secretion from dispersed salivary gland acini was shown to be specific for PGE 2, as compared with PGF 2α or the thromboxane analog U-46619, in accordance with their respective binding affinities for the PGE 2 receptor. Furthermore, the selective PGE 2 EP1 receptor agonist, 17-phenyl trinor PGE 2, was as effective as PGE 2 in stimulating secretion of anticoagulant protein. Calcium ionophore A-23187 (1 to 100 μM) stimulated secretion of anticoagulant protein in a dose-dependent manner but the voltage-gated Ca 2+-channel blocker verapamil (1 to 1000 μM) and the receptor-mediated Ca 2+-entry antagonist, SK&F 96365 (1 and 10 μM), and 5 mM ethylene glycol bis(β-aminoethyl ether)-N,N N′, N′-tetraacetic acid (EGTA) had no appreciable effect on inhibiting PGE 2-stimulated secretion of anticoagulant protein. PGE 2 (0.1 μM) and the non-hydrolyzable analog of guanosine triphosphate (GTP), GTPγS (10 μM), directly activated phospholipase C (PLC) in a membrane-enriched fraction of the salivary glands after PLC was first incubated with the PGE 2 EP1 receptor antagonist AH-6809, which presumably antagonized endogenous PGE 2 (0.3 μM) in the broken-cell-membrane-enriched fraction. TMB-8, an antagonist of intracellular inositol trisphosphate (IP 3) receptors, inhibited PGE 2-stimulated secretion. The results support the hypothesis that PGE 2 stimulates secretion of tick salivary gland protein via a phosphoinositide signaling pathway and mobilization of intracellular Ca 2+.

Molecular characterization of a Haemaphysalis longicornis tick salivary gland-associated 29-kilodalton protein and its effect as a vaccine against tick infestation in rabbits.

The use of tick vaccines in mammalian hosts has been shown to be the most promising alternative tick control method to current use of acaricides, which suffers from a number of limitations. However, the success of this method is dependent on the identification, cloning, and in vitro expression of tick molecules involved in the mediation of key physiological roles with respect to the biological success of a tick as a vector and pest. We have sequenced and characterized a Haemaphysalis longicornis tick salivary gland-associated cDNA coding for a 29-kDa extracellular matrix-like protein. This protein is expressed in both unfed and fed immature and mature H. longicornis ticks. The predicted amino acid sequence of p29 shows high homology to sequences of some known extracellular matrix like-proteins with the structural conservation similar to all known collagen proteins. Immunization with the recombinant p29 conferred a significant protective immunity in rabbits, resulting in reduced engorgement weight for adult ticks and up to 40 and 56% mortality in larvae and nymphs that fed on the immunized rabbits. We speculate that this protein is associated with formation of tick cement, a chemical compound that enables the tick to remain attached to the host, and suggest a role for p29 as a candidate tick vaccine molecule for the control of ticks. We have discussed our findings with respect to the search of tick molecules for vaccine candidates.

Kinetics and cross-species comparisons of host antibody responses to lone star ticks and American dog ticks (Acari: Ixodidae).

Understanding of the animal antibody response to tick salivary gland proteins is necessary to identify candidate antibodies that may have use as species- and feeding-duration-specific biomarkers of tick exposure in humans. The kinetics of the humoral immune response of rabbits to challenge feeding by 2 tick species [Amblyomma americanum (L.) and Dermacentor variabilis (Say)] was characterized by both enzyme-linked immunosorbent assay and immunoblot. Western blot analysis revealed that rabbits produced antibodies against both A. americanum and D. variabilis tick salivary gland antigens, with molecular weights ranging from 12.2 to 125 kDa; the antibody response against the saliva of both tick species possessed both unique and common aspects. The presence of antibody against low-molecular-mass (< 20 kDa) salivary gland antigens of A. americanum may be specific for A. americanum exposure. Antibodies directed against D. variabilis salivary gland antigens of 111, 86.3, and 85 kDa may be specific for D. variabilis exposure. The data suggest that host antibodies directed against specific tick salivary gland proteins might have use as species-specific biologic markers of tick exposure.

Tick Avoidance Behaviors Associated with a Decreased Risk of Anti-Tick Salivary Gland Protein Antibody Seropositivity in Military Personnel Exposed to Amblyomma Americanum in Arkansas

During April through September 1990, 399 military personnel who originated from either Fort Chaffee, Arkansas (n = 236) or Fort Wainwright, Alaska (n = 163) were studied during maneuvers in tick-infested areas at Fort Chaffee. Study subjects completed a questionnaire and had pre- and post-maneuvers serum specimens analyzed for antibodies to several rickettsial and ehrlichial agents and to Amblyomma americanum (lone star tick) salivary gland proteins (anti-tick saliva antibodies [ATSA], a biologic marker of tick exposure). Military rank/grade and home station were associated with pre-maneuvers ATSA seropositivity by enzyme-linked immunosorbent assay (ELISA). Fort Wainwright personnel were more likely to show at least a 50% increase in ATSA levels, compared with subjects from Fort Chaffee, from the pre- to the post-maneuvers specimen (adjusted odds ratio [AOR] = 2.7, 95% confidence interval [CI] = 1.2-6.1). Subjects from Fort Wainwright who did not report use of bed netting were at an increased risk of post-maneuvers ATSA seropositivity (AOR = 4.1, 95% CI = 1.5-11.5). In contrast, subjects from Fort Chaffee who did not report tucking pants into socks were at increased risk of post-maneuvers ATSA seropositivity (AOR = 2.8, 95% CI = 1.1-7.1). Subjects from Fort Chaffee who reported an attached tick bite during maneuvers were more likely to be ATSA-seropositive in the post-maneuvers specimen (AOR = 2.5, 95% CI = 1.2-5.2). Western blot assays revealed large differences in tick salivary gland proteins that were recognized on the post-maneuvers specimen among three randomly selected individuals, and small differences within a single individual who reported a tick bite during maneuvers, comparing pre- and post-maneuvers specimens. The ATSA ELISA seropositivity was not associated with seroconversion to the tick-borne infectious agents.

Salivary gland changes and host antibody responses associated with feeding of male lone star ticks (Acari:Ixodidae).

The goal of this study was to demonstrate that male lone star ticks, Amblyomma americanum (L.), actively feed on rabbits during attachment and that the host is capable of mounting an immune response against male salivary gland proteins. During attachment, it was shown that male ticks salivary glands hypertrophy. An enzyme-linked immunosorbent assay was used to detect rabbit serum proteins in the midgut of previously attached male ticks. SDS-polyacrylamide gel electrophoresis showed that the proteins expressed in the male tick salivary gland changed during feeding, with several new proteins in the 15- to 50-kDa range synthesized during attachment. Rabbits mounted a detectable antibody response against male tick salivary gland proteins after 2 sequential feedings of male ticks. The antibodies were directed against a spectrum of male salivary gland proteins ranging from 18 to 160 kDa. Several of these proteins were not recognized by antibodies directed against female tick saliva proteins, and thus may be specific male salivary gland components. This evidence indicates that male A. americanum ticks actively feed during attachment, that their salivary gland proteins change during feeding, and that male salivary gland proteins are immunogenic.

A secreted calreticulin protein in ixodid tick ( Amblyomma americanum) saliva

A complementary DNA clone from salivary glands of feeding female Amblyomma americanum ticks has been characterized as encoding calreticulin. Calreticulin, a major endoplasmic reticulum (ER) calcium-binding protein, appears to be secreted in Amblyomma and Dermacentor saliva. Evidence is accummulating that calreticulin performs roles unrelated to calcium storage. Unlike most known calreticulins, tick-secreted calreticulin lacks the ER retention signal, KDEL. This is the first molecular cloning of a specific tick salivary gland protein. The finding of a secreted calreticulin in tick saliva suggests a role for calreticulin in blood feeding through host immunosuppression or antihemostasis.